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Article by Nicole Rosas, Vanessa Li & Margaretha Morsink
miRNAs Promote Bone Regeneration after Radiation Therapy
Source Publication:
miR-34a promotes bone regeneration in irradiated bone defects by enhancing osteoblastic differentiation of mesenchymal stromal cells in rats, Stem Cell Research & Therapy, 2019
Huan Liu et al., Yimin Zhao Lab
Bone cancer is a serious and debilitating condition that affects both the structure and function of the skeletal system. It can cause severe pain and decline in mobility, significantly impacting a patient’s quality of life. Treatment often requires radiation therapy (radiotherapy) to shrink the tumor followed by surgical resection to remove the tumor. However, radiotherapy has a detrimental effect on bone’s healing ability, which can make reconstructive efforts, such as bone grafts, extremely difficult. To address these challenges, scientists have investigated the role of microRNAs, small molecules that help regulate gene activity and control how cells function. One specific microRNA, known as microRNA-34a (miR-34a), has been well-studied for its ability to suppress tumor growth. Recent studies have also uncovered its important role in bone biology, particularly in promoting the conversion of stem cells to osteoblasts, which help build bone. These findings suggest that miRNA-34a could serve as a potential treatment to enhance bone healing in patients undergoing radiation therapy.
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What did these researchers do?
MicroRNA are short, non-coding mRNA strands that do not make proteins, but help control gene expression. They attach to the messenger RNA (mRNA), which then either prevents it from turning into proteins or causes it to break down. In this study, researchers discovered that miR-34a is involved in turning certain stem cells, called bone marrow mesenchymal stromal cells (BMSCs), into osteoblasts. When they compared the levels of miR-34a in BMSCs from irradiated (damaged by radiation) and non-irradiated rat bones, they found more miR-34a in the irradiated cells. They also saw higher miR-34a levels in the newly formed bone in the irradiated group, suggesting that miR-34a helps the bone respond to radiation damage.
After establishing mirR-34a is involved in the bone formation process, the researchers tested what would happen if they added more miR-34a to the irradiated BMSCs. They found more alkaline phosphatase (ALP), a marker of bone cell activity, and increased calcium deposits, which showed that bone formation was happening more quickly. This was true even with different levels of radiation, suggesting that miR-34a improves the rate of bone healing after radiotherapy.
This procedure was repeated on mice that were exposed to radiotherapy and then underwent tumor resection surgery. These mice were treated with miR-34a and or an inhibitor to miR-34a, which was delivered via a collagen-based hydrogel. More regeneration was observed by delivering miR-34a compared to the negative control group, whereas less regenerated bone and a decreased bone volume was observed upon inhibition of miR-34a, indicating that the miRNA has a positive effect on the bone’s ability to heal after irradiation. To study how bones become stronger and form new tissue, they used a special fluorescent marker at different stages of the healing process. This helped scientists track how quickly and how much bone was growing back. They discovered that miR-34a helps speed up both processes, showing it could be useful for improving bone healing after radiotherapy.
Why is this important?
Radiotherapy is an essential step in the treatment of bone cancer as it effectively destroys malignant, dividing cancer cells and shrinks tumors. As a result, less bone needs to be removed, which helps keep more of the natural tissue intact. However, it also damages healthy adjacent tissue and complicates the bone’s healing response. These challenges could reduce reconstruction options after surgery and impact the patient’s quality of life. The discovery that miR-34a can enhance bone regeneration is a major advancement, offering a targeted approach to counteract these challenges caused by radiation. If translated into clinical practice, miR-34a therapy could transform outcomes for bone cancer patients undergoing radiotherapy. Beyond cancer treatment, this research also holds promise for addressing other conditions involving limited bone healing such as osteoporosis or trauma-induced bone defects.
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How did the researchers do this?
Bone marrow stem cells (BMSCs) were taken from 2-week-old rats and grown in the lab. These cells were then exposed to increasing amounts of X-ray radiation to mimic the effects of radiotherapy. Scientists studied the role of miR-34a in these cells using several techniques. To increase miR-34a levels, they used synthetic RNA mimics of miR-34a. To decrease its activity, they used inhibitors of miR-34a. These molecules were delivered into the cells using a method called RNA transfection, which introduces genetic material into cells to influence their behavior.
The researchers checked how the cells differentiated into bone-forming cells, called osteoblasts, using special tests. One test involved staining to measure mineral deposits where more intense staining meant more bone formation. They also used advanced qRT-PCR and Western Blotting to measure the levels of specific genes and proteins linked to bone formation, such as RUNX2, collagen type 1 (Col-1), and osteocalcin (OCN). RUNX2 helps cells become osteoblasts, Col-1 forms the bone structure, and OCN indicates mature bone-forming cells.
For the in vivo (live animal) experiment, researchers created small 3-mm holes in the leg bones of irradiated rats to study bone healing. They used collagen hydrogels to deliver miR-34a directly to the injury site. The hydrogel acts like a soft scaffold, helping to hold the treatment in place and focus it where it’s needed. To check how well the bone was healing, they used a technique called micro-CT. This created detailed 3D images of the bone and allowed them to measure how much new bone formed compared to the total bone volume (BV/TV). Special stains were also used to visualize the bone structure, and sequential labeling helped track the different stages of bone formation over time.

Assessment of bone structure and regeneration with addition of miRNA34a (agomiR-34a) or inhibition of miRNA-34a (antagomiR-34a) in bones that have been irradiated
What comes next?
While this study shows that miR-34a could help with bone healing, its effects on other cells involved in the process haven’t been fully explored. For example, miR-34a has been found to slow down the activity of cells that break down bone, called osteoclasts, and it may also block the growth of blood vessels in bones, which are crucial for healing. This means scientists need to find ways to target specific cells to avoid unwanted side effects and make the treatment safe and effective. The study used a collagen-based hydrogel to deliver miR-34a, which allowed for controlled release. However, the release profile of the miRNA delivery needs further investigation to determine if it provides sustained delivery or would require multiple injections. Additionally, this research was done over a short period. Future studies should evaluate the long-term effects of miR-34 therapy as well as assess the biomechanical properties of the regenerated bone.